Factor V plays a pivotal role in hemostasis, and in the pathology of venous thrombosis. Fluorescence-based equilibrium binding, steady-state and rapid reaction kinetics are proposed to define the molecular mechanisms of the procoagulant activation of human factor V, factor Va cofactor activity in regulating prothrombin (ProT) activation, and the mechanism of the anticoagulant inactivation of factor Va by activated protein C (ARC). The broad goal is an understanding of the interdependence of these mechanisms in hemostasis and thrombotic disease. Aim 1 will test the hypothesis that factor V activation by thrombin and meizothrombin (MzT) involves exosite I and ll-mediated substrate recognition of factor V and its activation intermediates that order the pathway and control the generation of specific binding sites for factor Xa and ProT. Aim 2 will evaluate the hypothesis that factor Va cofactor activity involves a two-step mechanism of substrate recognition of ProT and its activation intermediates, with initial formation of a complex in which the factor Xa catalytic site is accessible, followed by conformational engagement of the catalytic site. (Pro)exosite I on ProT substrate species is postulated to control one or both steps in substrate recognition through interactions with factor Va in the prothrombinase complex. Aim 3 will elucidate the mechanism of ordered cleavage of the ProT activation pathway hypothesized to result from ProT binding in two alternate conformations that present the Arg320 and Arg271 cleavage sites sequentially to factor Xa. Differential (pro)exosite I expression accompanying ProT activation is postulated to switch ProT between these conformations, thereby controlling the pathway. Aim 4 will test the hypothesis that regulation of the rate and pathway of factor Va inactivation by APC through ordered cleavages at Arg506, Arg306, and Arg679 is achieved by exosite-mediated competitive binding of factor Xa and APC to factor Va for cleavage at Arg506, and specific acceleration of cleavage at Arg306 by protein S. APC-factor Va substrate recognition is postulated to follow the two-step mechanism in which factor Xa and protein S switch APC between alternate bound conformations to direct cleavage at Arg506 and Arg306. New knowledge of exosite interactions in the physiological substrate specificity of coagulation proteinases will facilitate the development of novel exosite-directed anticoagulants. Relevance. Blood clots occurring abnormally in the deep veins of the legs can break apart in the circulation and lodge in the lungs, resulting in life-threatening thrombosis. Individuals with fairly common genetic defects in the coagulation factors, factor V, protein C, protein S, and prothrombin, and women taking oral contraceptives have a higher risk of clotting in veins. The goal of the research is to understand how these proteins work in normal clotting and in clotting diseases. The results may lead to new drugs for treatment of clotting diseases.